The broad, long-term objective of this research plan is a better understanding of the structural and catalytic repertoire of RNA. The focus is on a set of at least 65 RNA catalysts (ribozymes) that have been isolated from random RNA sequences by means of recently developed molecular biology techniques called in vitro selection and in vitro evolution. The new ribozymes catalyze RNA ligation reactions, and based on ligation regioselectivities and substrate-binding modes these ligases can be grouped into at least three classes. The specific aims of this proposal are: (1) To characterize catalytic and structural features of the three classes of ligases. Structural features will be examined with molecular biology methods including modification interference, substitution interference, and in vitro selection from degenerate sequence pools. Candidate molecules for crystallization will also be designed and screened. The catalytic mechanisms of the three ligase classes will be probed by examining the effects of thio substitutions at the reactive phosphate, the pH dependence of the reactions, and the effects of substituting Mg2+ with alternative divalent cation cofactors. (2) To extend the known repertoire of RNA catalysis by generating ligases with polymerase-like substrate-binding properties. The selected pool of >65 ligases will serve as starting molecules for in vitro selection and evolution procedures designed to isolate and evolve ligases that utilize sugar-phosphate backbone contacts for substrate recognition. Because the new ribozyme classes comprise a significant fraction of the known RNA catalytic motifs, the detailed characterization of these Iigases will contribute to basic knowledge of RNA molecular biology. Understanding of natural RNA structures and catalysts will be enhanced when put into the broader context of what is possible when liberated from the constraints of natural evolution. In addition to further extending the known range of RNA catalysis, isolating ligases with polymerase-like properties will speak to the ability of RNA to catalyze self-replication - a central supposition of current theories of the early evolution of life. Characterizing the three new classes of ligases and isolating additional ligases with the demanding substrate-binding properties of a polymerase will provide insights into the potential, the limitations, and possible improvements for the in vitro selection technology. Such insights, when combined with a better understanding of the scope of reactions that RNA is capable of catalyzing, will be useful for those attempting to exploit the selection technology to generate new pharmaceuticals and other beneficial molecules.